This invention relates to methods for the chemical modification of mammal urine and mammal blood. The modified blood or the modified urine have immune modulatory effects when administered to a mammal and can be used for the treatment of immune disorders.
Mammal blood is known to consist of a cellular and a non cellular fraction. The non cellular fraction in non hemolytic blood is called plasma, a watery solution of minerals and complex biochemical synthesis products such as proteins (derived mostly from the liver and in form of immuneglobulines from cells of the immune system), carbohydrates, proteoglycans etc.
The cellular phase can be separated by centrifugation or spontaneous sedimentation of the blood cell components and yields mostly erythrocytes and thrombocytes and a smaller fraction of leukocytes. Those can be differentiated in granulocytes and lymphocytes. Newer research has turned especially to the subgroups of the lymphocytes through flow cytometry. After specific staining of surface antigens it has been possible to identify different groups of cells among these. Cells with CD4 were called T-Helper cells (inducing and increasing immune activity), CD8 Cells were called T supressor or cytotoxic cells (reducing and controlling immune activity). A further discrimination of T helper cells in Th1 and Th2 cells was possible. Th1 cells produce preferential proinflammatory cytokins like interleukin 2, interferon gamma and tumor necrosis factor. Th1 cells are used for the attack of intacellular target antigens and tumor cells by their cytotoxic activity. The action of Th2 cells is preferential anti-inflammatory, they are producing Interleukin (IL) 4, IL 10 and IL 13. Th2 cells are used for the control of extracellular antigens. By the interaction of the Th1, the Th2 and the antigen presenting cells, the effector action of the immune system is controlled. Interferon gamma enhances the production of proinflammatory substances as tumor necrosis factor, IL 1, IL 6, IL 12, oxygen radicals and NO. IL 4 is counteracting the proinflammatory activity of these substances. Different groups of diseases show typical shifts of the balance in favor of Th1 or Th2 cells. A shift in favor of the Th1 cells is considered positive for tumor patients (anti tumor activity) but noxious for patients with organ specific autoimmune diseases (like rheumatoid arthritis, uveitis, thyreoiditis). A predominance of Th2 cells will be noxious for allergic disease (Eczema), immune globulin mediated diseases and sclerodermia. This is considered as a possible explanation for the fact, that patients with active allergies and neurodermatitis are less prone for cancer. The pathologic activity and overweight of parts of the immune system in patients with allergic skin or lung disease, rheumatic and other autoimmune disease as well as many types of cancer has led to search for complex immunemodulatory agents that can rebalance the causing unbalance to influence the resulting disease.
Mammal urine or alternatively ultrafiltrate of dialysis treatments contain a spectrum of proteins and proteoglycans and other substances that pass physiologically the kidney filter. The substances are complex and differ in size and biologic function. A lot of urine substances probably have a biologic effect, but the subcutaneous administration of sterile urine of the same host mammal has not shown mentionable effects on the cells of the immune system. The same applies to the addition of sterile urine to leukocyte cultures of the same individual, no remarkable immunemodulatory effect was observed.
U.S. Pat. No. 4,632,980 (Zee et al.) discloses a method for virus inactivation of blood and blood products comprising treatment with low levels of ozone. After virus inactivation the blood components are further separated and it can be used for the intended purpose.
U.S. Pat. No. 4,684,521 (Edelson) discloses a method and system for externally treating blood. It comprises treatment of blood with UV radiation in the presence of a photoactive agent.
U.S. Pat. No. 4,748,120 (Wiesehahn) discloses a method for the treatment of biological compositions with psoralen derivatives under irradiation conditions.
It has now been found that oxidation of blood and urine under special conditions yield a reliable substance for the treatment of disorders of the immune system. In one embodiment the present invention provides a method for chemical modification of mammal urine comprising the steps of
collecting urine from a mammal,
treating said mammal urine with an oxidizing agent and a gas atmosphere of at least 90% to 100% (v/v) oxygen in a container,
adding at least one protease,
removing substances with a low molecular weight to yield a modified mammal urine.
In a second embodiment the present invention provides a method for chemical modification of mammal blood comprising the steps of
collecting blood from a mammal,
separating the blood in a plasma phase and a cell phase,
treating of the plasma phase, the cell phase or both with an oxidizing agent and a gas atmosphere of about 90% to 100% (v/v) oxygen in a container,
combining the plasma phase with the cell phase,
adding a cell culture medium and at least one protease,
adding modified mammal urine prepared according to the first embodiment,
incubating for 16 to 36 hours at about 37xc2x0 C.,
adding a preservation agent to yield modified mammal blood.
In a third embodiment the invention provides a method of treating immune disorders comprising the step of administering to a patient in need thereof modified mammal urine prepared according to the method of the invention.
In a fourth embodiment the invention provides a method of treating immune disorders comprising the step of administering to a patient in need thereof modified mammal blood prepared according to the method of the invention.
In a fifth embodiment the present invention provides the substance being able to modulate the immune system.
The first embodiment of the present invention discloses a method for the chemical modification of mammal urine. This method comprises the step of collecting urine from mammal. It is preferred that the collection is performed in a way to enable sterile collection of the urine. This can be done by sterile cathederization of the urinary bladder or collection of midstream urine. To keep the urine sterile a sterile container should be used, e.g. a disposable sterile plastic container. Depending on the presence of non soluble components in the urine the urine can be centrifuged and the sediment may be discarded. Conventional test methods for the screening of bacterial contaminations, are e.g. sticks test on nitrite.
The collected mammal urine is now treated with an oxidizing agent at a gas atmosphere of at least about 90% (v/v) oxygen. The term xe2x80x9coxygenxe2x80x9d is meant to comprise the clement oxygen in atomic and molecular pure forms, especially in the form of O2 and O3. O2 is the main component of the gas atmosphere. As oxidizing agents H2O2 and O3 are especially preferred. H2O2 may be added as a diluted solution to the urine. Suitable amounts are in the range of 1 to 3 ml of a one percent solution of H2O2 per 100 ml of urine. The ozone (O3) may be added to the gas atmosphere.
If ozone is used as an oxidizing agent it should be present in a concentration of about 50 to 100 xcexcg/ml of the oxygen atmosphere. Apparatus for the generation of ozone are commercially available and are described, e.g. in U.S. Pat. No. 5,052,382 to Waenwright and U.S. Pat. No. 5,053,140 to Horste, both incorporated by reference. It is of high importance to perform the oxidation in a gas atmosphere having a concentration of at least 90% (v/v) of O2. The inventors of the present invention have revealed that his combination of weak oxidizing O2 with a strong oxidizing agent allows a reliable and reproducible modification of mammal urine. It is preferred that the O2 concentration in the gas atmosphere is as high as possible, preferably at least 95% and more preferably at least 99%. It is most preferred that the concentration of O2 is as high as commercially available. The only relevant other gas may be ozone in the case that ozone is used as an oxidizing agent. The oxygen should be of medical grade to avoid contaminations of the urine. The oxygen atmosphere in the container is obtained by removing the air atmosphere above the urine in the container or use of an evacuated container and addition of the O2 atmosphere. The O2 atmosphere may have a pressure in the range of less than atmospheric pressure up to about 1 bar above atmospheric pressure. It is preferred that the pressure in the container is close to atmospheric pressure because this facilitates handling and avoids complicated mechanisms for tight scaling of the container. It is preferred to provide intimate contact between the oxygen atmosphere and the urine. A suitable method is shaking of the urine with the oxygen atmosphere to allow reaction between the urine, the oxidizing agent and the oxygen atmosphere. The concentration of the oxidizing agent may be varied in a broad range which influences certainly the reaction velocity of the oxidizing agent. Suitable concentrations of the oxidizing agent are in a range of 1 to 5 xcexcmole per ml urine. It is obvious for a person skilled in the art that the oxidizing agent should not incorporate any toxic substances into the urine. To allow intimate mixture of the gas atmosphere with the urine it is preferred that the volume of the gas atmosphere is at least 50% of the volume of the urine. Preferably, it is in the range of 50 to 200% of the volume of the urine and more preferably of about the same volume as the urine.
After adding the oxidizing agent at least one protease must be added. Suitable proteases comprise serine proteases such as papaine, trpysine, chymotrypsine, mixtures thereof and the like. The enzymatic activity should be in the range of 0.1 to 2 microkat per milliliter of urine. One kat is defined as the amount of an enzyme cutting one mole of peptide bonds per second. After the addition of the oxidizing agent and the at least one protease the urine is incubated in the dark in a range of 2 to 18 hours at a temperature of preferably 37xc2x0 C. Lower temperatures may be used but this may require increasing the amount of oxidizing agent and the amount of proteases or extending the incubation time because the reaction velocity depends on the temperature and the concentration of the reagents. Slightly higher temperatures may also be used but substantially higher temperatures may denature sensitive components of the urine and may decrease the efficiency of the modified urine.
After the incubation, substances with a low molecular weight should be removed. The term xe2x80x9csubstances with a low molecular weightxe2x80x9d is used to define substances having a molecular weight below about 6,000 Dalton, more preferably below about 5,000 Dalton. A preferred method for the removal is the use of an ultrafiltration unit. Suitable ultrafiltration membranes are, e.g. Ultrafree-15-Biomac-5K (commercially available from Milipore Inc. The ultrafiltration can be conveniently performed with the aid of a centrifuge which speeds up the process of ultrafiltration. Ultrafiltration furthermore reduces the amount of water in the urine thus increases the concentration of the high molecular weight components, Instead of ultrafiltration other methods can be used. As suitable method is e.g. gel filtration. After the removal of the low molecular weight substances and optionally water, the modified mammal urine is preferably treated again with an oxidizing agent and a gas atmosphere as described above. This repeated treatment increases the share of the components which are modified by the method of the present invention and thus increases the effectiveness of the treatment. The obtained modified mammal urine should be stored below 4xc2x0 C. or may be frozen.
As a further embodiment the present invention discloses a method for chemical modification of mammal blood comprising treating the blood with the modified mammal urine prepared according to the first embodiment. The method comprises the step of collecting blood from a mammal. This blood is collected in the usual medical way and is transferred into the container comprising heparine to avoid clotting of the blood. 6,000 IU sterile sodium heparine (volume 2 to 5 ml) or the equivalent amount of low molecular heparine are sufficient for about 200 ml blood. Alternatively, a bone marrow aspirate or homogenized tissue containing leukocytes can be used. As described above sterile conditions should be met during collecting and further processing of the blood. Laminar flow conditions are preferably used during further processing. A sample of the collected blood is checked for HIV, hepatitis B and C in the usual way to warrant safety of the laboratory personal.
The blood is then separated in a plasma phase and a cell phase. This can be done by centrifugation at approximately 750 g or alternatively by allowing the cell compartment (comprising erythrocytes, thrombocytes and leukocytes) to sediment in the container.
After separation of the cell phase and the plasma phase into containers the plasma phase, the cell phase or both are treated with an oxidizing agent and a gas atmosphere of at least 90% (v/v) oxygen. The description of the oxidation step above equally apply to the treatment of the plasma phase and the cell phase.
Without wishing to be bound to a theory it is believed that the leukocytes proliferate as a result of the treatment with the oxidizing conditions or increase production of immunogenic products. To enable proliferation, a cell culture medium is added which comprises salts and amino acids as routinely used in cell culture. Suitable media are commercially available. A preferred, but not limiting example of a cell culture medium is described in the examples. A 4.2% (v/v) NaHCO3 solution can be added to adjust the pH. Furthermore, a suitable amount of at least one protease is added. Suitable proteases and amounts are explained above.
Thereafter, modified mammal urine prepared according to the first embodiment of the invention, preferably from the same mammal is added. This mixture is incubated for about of 16 to 36 hours at about 30xc2x0 C. in the darkness. A conventional role mixer or shaker can be used. Significantly higher or lower temperature conditions would strongly influence the proliferation efficiency of the cells. It is, therefore, considered important to keep the temperature in the range from 35 to 40xc2x0 C. It is believed that under this conditions the cells start to proliferate or produce a disease specific substance pattern. It is believed that the leukocytes of a person with an immune disorder contain specific information for the production of a specific remedy or for the production of the remedy inducing function if administered to the donor of the leukocytes for this disorder. After the incubation a suitable preservation agent is added. A number of preservation agents for the preservation of blood are known. Suitable solutions are known under the abbreviations ACD (anticoagulant citrate/dextrose solution) and CPD (anticoagulant citrate phosphate dextrose solution). These solutions comprise a citrate puffer system, glucose and optionally a sodium phosphate salt. These preservation agents have a weak acidic pH between 4 and 6. A suitable preservative premix is ACD stabilizator Fon A commercially available from Fresenius, Germany.
The modified urine and the modified blood of the present invention may be used for treating immune disorders. Allergic diseases, rheumatic diseases, autoimmune diseases and immune deficient diseases can be successfully treated or at least be improved by this treatments. Allergic diseases include e.g. Hay fever (rhinoconjunctivitis allergica, allergic asthma, neurodermitis (Eczema) etc. Rheumatic diseases include e.g. chronic polyarthritis, systemic lupus erythermatodes etc. Other autoimmune diseases include immune vasculites, immune nephritis etc. Immune deficiencies include e.g. acquired immune deficiencies, chronic virus infections. Before administration the person skilled in the art would be aware that it is necessary to check the modified urine or the modified blood for sterility and absence of endotoxins. Suitable tests comprise conventional medical urine in cultures, e.g. uricult or conventional medical blood culture. Absence of endotoxins can be checked with Limulus Amebocyte Lysate Test (BioWhittakker Inc., Walkersville, USA). The substances prepared according to the method of the invention can be administered orally, transdermally, by inhalation or parentally. It is preferred that the modified blood or the modified urine are prepared from the urine or the blood of the patient to be treated with the modified urine or the modified blood.
In a further embodiment the present invention provides substances which have an immune modulatory effect. Treating of urine from patients with, e.g. severe allergic diseases show the disappearance of two peaks and the appearance of two new peaks. This effect is not seen in healthy individuals. It is believed that these new isolated substances can be isolated and may be successfully used as immune modulatory substances.